The induction of antigen-specific T cell responses involves multiple interactions between cell surface receptors on T cells and ligands on antigen presenting cells (APCs). The primary interaction is between the T cell receptor (TCR)/CD3 complex on a T cell and a major histocompatibility complex (MHC) molecule/antigenic peptide complex on an antigen presenting cell. This interaction triggers a primary, antigen-specific, activation signal in the T cell. In addition to the primary activation signal, induction of T cell responses requires a second, costimulatory signal. In the absence of proper costimulation, TCR signaling can induce a state of anergy in the T cell. Subsequent appropriate presentation of antigen to an anergic T cell fails to elicit a proper response (see Schwartz, R. H. (1990) Science 248:1349).
A costimulatory signal can be triggered in a T cell through a T cell surface receptor, such as CD28. For example, it has been demonstrated that suboptimal polyclonal stimulation of T cells (e.g. by anti-CD3 antibodies or phorbol ester, either of which can provide a primary activation signal) can be potentiated by crosslinking of CD28 with anti-CD28 antibodies (Linsley, P. S. et al. (1991) J. Exp. Med. 173:721; Gimmi, C. D. et al. (1991) Proc. Natl. Acad. Sci. USA 88:6575). Moreover, stimulation of CD28 can prevent the induction of anergy in T cell clones (Harding, F. A. (1992) Nature 356:607-609). Natural ligands for CD28 have been identified on APCs. CD28 ligands include members of the B7 family of proteins, such as B7-1(CD80) and B7-2 (B70) (Freedman, A. S. et al. (1987) J. Immunol. 137:3260-3267; Freeman, G. J. et al. (1989) J. Immunol. 143:2714-2722; Freeman, G. J. et al. (1991) J. Exp. Med. 174:625-631; Freeman, G. J. et al. (1993) Science 26:909-911; Azuma, M. et al. (1993) Nature 366:76-79; Freeman, G. J. et al. (1993) J. Exp. Med. 178:2185-2192). In addition to CD28, proteins of the B7 family have been shown to bind another surface receptor on T cells related to CD28, termed CTLA4, which may also play a role in T cell costimulation (Linsley, P. S. (1991) J. Exp. Med. 174:561-569; Freeman, G. J. et al. (1993) Science 262:909-911).
The elucidation of the receptor:ligand relationship of CD28/CTLA4 and the B7 family of proteins, and the role of this interaction in costimulation, has led to therapeutic approaches involving manipulation of the extracellular interactions of surface receptors on T cells which bind costimulatory molecules. For example, a CTLA4Ig fusion protein, which binds to both B7-1 and B7-2 and blocks their interaction with CD28/CTLA4, has been used to inhibit rejection of allogeneic and xenogeneic grafts (see e.g., Turka, L. A. et al. (1992) Proc. Natl. Acad. Sci. USA 89:11102-11105; Lenschow, D. J. et al. (1992) Science 257:789-792). Similarly, antibodies reactive with B7-1 and/or B7-2 have been used to inhibit T cell proliferation and IL-2 production in vitro and inhibit primary immune responses to antigen in vivo (Hathcock K. S. et al. (1993) Science 262:905-907; Azuma, M. et al. (1993) Nature 366:76-79; Powers, G. D. et al. (1994) Cell. Immunol. 153:298-311; Chen C. et al. (1994) J. Immunol. 152:2105-2114). Together, these studies indicate that T cell surface receptors which bind costimulatory molecules such as B7-1 and B7-2 are desirable targets for manipulating immune responses.
While the extracellular interactions between CD28/CTLA4 with their ligands have been characterized in some detail, little is known regarding the intracellular events that occur in a T cell following ligation of these molecules. T cell costimulation is thought to involve an intracellular signal transduction pathway distinct from signalling through the TCR since the costimulatory pathway is resistant to the inhibitory effects of cyclosporin A (see June, C. H. et al. (1990) Immunology Today 11:211-216) Protein tyrosine phosphorylation has been shown to occur in T cells upon CD28 ligation and it has been demonstrated that a protein tyrosine kinase inhibitor, herbimycin A, can inhibit CD28-induced IL-2 production (Vandenberghe, P. et al. (1992) J. Exp. Med. 175:951-960; Lu, Y. et al. (1992) J. Immunol. 149:24-29). 
This invention relates to the regulation of T cell responses by manipulation of intracellular signal transduction. In particular, intracellulal signalling events which occur upon costimulation of a T cell are manipulated. The invention encompasses methods for inhibiting or stimulating T cell responses by inhibiting or stimulating one or more intracellular signals which result from ligation of a surface receptor on a T cell which binds a costimulatory molecule. It has now been discovered that CD28 receptor stimulation leads to the production of D3-phosphoinositides within a T cell. Moreover, it has been discovered that inhibition of the activity of phosphatidylinositol 3-kinase in a T cell can inhibit T cell responses, such as lymphokine production and cellular proliferation. These discoveries indicate a functional role for D3-phosphoinositides in a costimulatory signal transduction pathway and provide phosphatidylinositol 3-kinase as an intracellular target for modulation of T cell responses. Accordingly, intracellular signalling events involving D3-phosphoinositides can be modulated either to inhibit a costimulatory signal and thereby induce T cell unresponsiveness, or to trigger a costimulatory signal and thereby generate a T cell response. In addition, novel screening assays for identifying inhibitors or activators of phosphatidylinositol 3-kinase, which can be used to inhibit or stimulate a T cell response, are within the scope of the invention.
One aspect of the invention pertains to methods for inhibiting a response by a T cell which expresses a surface receptor that binds a costimulatory molecule. These methods involve contacting the T cell with an agent which inhibits production of D3-phosphoinositides in the T cell. In one embodiment, the agent is an inhibitor of a phosphatidylinositol 3-kinase, such as the fungal metabolite wortmannin or the bioflavenoid quercetin, or derivatives or analogues thereof (e.g. LY294002). In another embodiment of the method of the invention, the T cell is contacted with at least one additional agent which inhibits a different intracellular signal associated with costimulation, such as protein tyrosine phosphorylation. For example, the T cell can be contacted both with an inhibitor of phosphatidylinositol 3-kinase and with an inhibitor of a protein tyrosine kinase. A preferred inhibitor of a protein tyrosine kinase is herbimycin A. Alternatively, protein tyrosine phosphorylation can be inhibited in a T cell by a tyrosine phosphatase or an activator of a tyrosine phosphatase. In this embodiment, the T cell can be contacted with an inhibitor of phosphatidylinositol 3-kinase and with a molecule, e.g., an antibody, which binds to and activates a cellular tyrosine phosphatase, such as CD45 or Hcph.
The invention also provides methods for inducing unresponsiveness to an antigen in a T cell by triggering a primary, antigen-specific signal in a T cell while interfering with an intracellular signal associated with costimulation in the T cell. As a result of interfering with costimulatory signal transduction, the T cell fails to receive a proper costimulatory signal in the presence of the antigen and antigen-specific unresponsiveness is induced in the T cell. To induce T cell unresponsiveness, an antigen-specific T cell is contacted with the antigen in a form suitable for stimulation of a primary activation signal in the T cell, together with an agent which inhibits production of D-3 phosphoinositides in the T cell. For example, a T cell can be contacted with an antigen presented by an APC together with an inhibitor of phosphatidylinositol 3-kinase, such as wortmannin or quercetin or derivatives or analogues thereof (e.g. LY294002). Additionally, other intracellular signals associated with costimulation, such as protein tyrosine phosphorylation, can be inhibited in the T cell.
Methods for inhibiting T cell responses and for inducing T cell unresponsiveness are useful in situations where it is desirable to down-modulate an immune response, for example in a transplant recipient (e.g., of an organ graft or bone marrow graft etc.) or a subject suffering from an autoimmune disease or other disorder associated with an abnormal immune response. An agent which inhibits signal transduction associated with costimulation (e.g., an inhibitor of inositol phosphate 3-kinase) can be administered to a subject or, alternatively, T cells can be obtained from the subject, treated in vitro as described herein and administered to the subject.
Another aspect of the invention pertains to methods for stimulating a response by a T cell which has received a primary activation signal and expresses a surface receptor that binds a costimulatory molecule. These methods involve contacting the T cell with an agent which stimulates production of D-3 phosphoinositides in the T cell, such as an activator of phosphatidylinositol 3-kinase. In another embodiment, the T cell is contacted with an agent which stimulates production of D-3 phosphoinositides and at least one additional agent which stimulates a different intracellular signal associated with costimulation, such as protein tyrosine phosphorylation. For example, the T cell can be contacted with an activator of phosphatidylinositol 3-kinase together with an activator of a protein tyrosine kinase, such as pervanadate. Alternatively, an inhibitor of a cellular phosphatase, such as CD45 or Hcph, can be used in conjunction with a PI3K activator. In yet another embodiment of the invention, an antigen-specific T cell response is stimulated by contacting an antigen-specific T cell with the antigen together with an agent which stimulates production of D-3 phosphoinositides in the T cell, thereby stimulating both a primary activation signal and a costimulatory signal in the T cell.
Methods for stimulating T cell responses are useful in situations where it is desirable to up-regulate an immune response. For example a response against a tumor in a tumor-bearing subject can be stimulated or a response against a pathogen (e.g., a bacteria, a virus, such as HIV, fungus, parasite etc.) in a subject can be stimulated. Additionally, the methods can be used to enhance the efficacy of vaccination. An agent which stimulates an intracellular signal associated with costimulation (e.g., an activator of inositol phosphate 3-kinase) can be administered to a subject or, alternatively, T cells can be stimulated in vitro and then administered to a subject.
Another aspect of the invention pertains to screening assays for identifying inhibitors or activators of a phosphatidylinositol 3-kinase. In one embodiment, a T cell which expresses a cell surface receptor (e.g., CD28) which binds a costimulatory molecule is utilized. To identify an inhibitor, an intracellular signal transduction pathway associated with the receptor in the T cell is stimulated in the presence of an agent to be tested and an inhibitor is identified based upon its ability inhibit production of at least one D-3 phosphoinositide in a T cell. To identify an activator, the T cell is contacted with an agent to be tested and an activator is identified based upon its ability to stimulate production of at least one D-3 phosphoinositide in a T cell.